Selective Delivery of Material to Cells

ABSTRACT

Isolating or identifying a cell based on a physical property of said cell can include providing a cell suspension; passing said suspension through a microfluidic channel that includes a constriction; passing the cell suspension through the constriction; and, contacting said cell suspension solution with a compound. The constriction can be sized to preferentially deform a relatively larger cell compared to a relatively smaller cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/866,972 filed Aug. 16, 2013, the entire contents of which ishereby expressly incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under R01GM101420-01A1awarded by the National Institutes of Health, RC1 EB011187-02 awarded bythe National Institutes of Health, P30-CA-14051 awarded by the NationalCancer Institute, and GRFP Primary Award number is #1122374 awarded bythe National Science Foundation. The Government has certain rights inthe invention.

TECHNICAL FIELD

The field of the invention relates to size-selective delivery ofmaterial to cells.

BACKGROUND

Intracellular delivery of materials is a challenge. Existingtechnologies that rely on nanoparticles, electrical fields, pore-formingchemicals, etc. are capable of delivering some materials to certain celltypes but often in an indiscriminant fashion with regards to thephysical properties of the target cell. By developing selective deliverymethods dependent on the physical properties of the target cells, onecould exert more robust control in delivery activity for research,diagnostic or therapeutic applications. For example, Circulating tumorcells (CTCs) are tumor cells found in the bloodstream, believed tomediate metastasis, or the spread of cancer, to distant sites in thebody. Approximately 90% of human deaths from cancer are due tometastasis. Identification and characterization of CTCs could be the keyto understanding, treating, or preventing metastatic cancer. Moreoverthese cells are known to have different physical properties compared tothe surrounding blood cells.

SUMMARY

The current subject matter provides devices, systems, and methods forselectively delivering material to one or more cells based on theirphysical properties, such as size, volume, diameter, cytosol viscosity,or membrane stiffness. For example materials can be delivered in a cellsize dependent manner. A cell suspension containing differentially sizedcells can be run through a device in the presence of the target deliverymaterial (e.g., a dye, a protein, nucleic acid, and the like) and thesematerials can be selectively delivered to the larger cells within thepopulation. The mechanism of delivery in the data being throughselective disruption of the cell membrane of larger cells as they aredeformed in a channel constriction while smaller cells are not deformedenough to cause membrane disruption.

In some example implementations, labelling tumor cells relative tonon-tumor cells can be achieved. Cells are run through a device for sizeselective tagging using fluorescent dyes or other detectable markers.The cells are optionally stained with an antibody, e.g., a tumor cellselective antibody, e.g., antibodies against CD45 to provide furthercontrast between cancer cells and blood cells (most blood cells areCD45+). The samples are run through a cell sorter, e.g. a standardfluorescence-activated cell sorter (FACS).

In some example implementations, labeling of cells based on their cellcycle can be achieved because cells within a population that are closerto division are larger than those that have just undergone division.Delivery of a dye to the bigger cells within a population can be used toidentify the individual cells that are in a later stage of their cellcycle.

In some example implementations, therapeutics for blood cancers (e.g.lymphomas) can be achieved because lymphoma cells are often bigger thanthe surrounding blood cells thus an intracellular toxin can be deliveredto lymphoma cells but not the healthy surrounding blood cells. This caninduce selective death of diseased cells.

Tagged cells can be isolated by fluorescence or magnetic purificationtechniques. Flow cytometry or microarrays with robotic manipulators canbe used to select cells based on fluorescence, while magnetic columns,microfluidic magnetic separation systems, or magnetic sweepers can beused to isolate magnetically tagged particles.

Cells can be identified based on relative size or diameter. Thus,relatively larger cells selectively or preferentially take up markers,because the extent of cell membrane disruption is relatively greater inlarger cells, i.e., larger cells are deformed to a greater extentcompared to smaller cells. Due to the greater degree of membranedisruption of larger cells, at least 10%, 25%, 50%, 2-fold, 5-fold,10-fold, 100-fold or more of a payload molecule gains access to theinside (cytoplasm) of a larger cell compared to a smaller cell. As aresult of the uptake of detectable markers in this manner and subsequentsorting based on uptake of the marker, the purity of tumor cells isenhanced by 100 times; 1,000 times, and up to 10,000 times or morecompared to the level of purity in peripheral blood. Purity is assessedby an antibody that targets/binds to a known marker that isexpressed/overexpressed by tumor cells. Alternatively, antibodiesagainst markers that are not expressed by tumor cells but areexpressed/overexpressed by blood cells (CD45 is an example). Eitherapproach helps provide increased contrast to sort out the cells ofinterest.

Samples with high size-tag fluorescence and low CD45 fluorescence arecaptured as candidate/potential CTCs. FACS outputs are inherentlyrelative. A “high” signal is minimum one decade (ten times higher level)of fluorescence intensity above the baseline control signal, and a “low”is one decade below the positive control population.

The device and methods of the invention provide a solution to thelong-standing problem of how to identify and/or isolate approximatelyfor more (2, 5, 10, 100, 1,000 or more) CTCs per 1-10 million leukocytesin a patient-derived sample of blood. For example, 1 CTC per ml of bloodis clinically relevant in a cancer patient. Accordingly, a method forisolating or identifying a circulating tumor cell comprises the steps ofproviding a cell suspension; passing the solution through a microfluidicchannel that includes a constriction, the constriction being sized topreferentially deform a circulating tumor cell compared to a leukocyte;passing the cell suspension through the constriction; and contacting thecell suspension solution with a detectable marker. The suspension can bepassed through a microfluidic channel that includes a constriction, theconstriction being sized to preferentially deliver a compound to a groupof cells having a relatively different physical property than anothergroup of cells. The physical property can include cell size, diameter,cytosol viscosity, and/or membrane stiffness (e.g., as measured bytransit time assays, stiffer cells pass through specializedmicrochannels more slowly than less stiff cells, e.g., as described inSharei et al., 2012, Anal. Chem. 84(15):6438-6443; Cross et al., 2007,Nature Nanotechnology 2:780-783). The contact can happen afterdeformation treatment. Or the material can be premixed with the cellsbefore deformation treatment. Both CTCs and leukocytes are deformed;however larger cells are deformed to a greater degree and therefore,molecules are selectively delivered to such cells, thereby treating ortagging them.

For example, the marker comprises a detectably labeled, e.g.,fluorescently or magnetically labeled material, such as a dye orparticle. The dyes or particles need not be tumor specific. Optionally,they differentially bind to tumor cells (e.g., at least 20%, 50%, 2times, 5 times, or more compared to non-tumor cells). However, thespecificity of the method is based on the discovery that tumor cells areslightly larger than leukocytes and the device is highly size selective.This size difference depends on the tumor type. For example, tumor cellsare generally from 50%-400% larger than the leukocytes. Therefore, thedelivery material preferentially enters into cells that are large enoughto be tagged via size-specific deformation of cells. The delivered tagis then in turn detected to identify the CTC.

In one example, the suspension comprises whole blood. Alternatively, thecell suspension is a mixture of cells in a physiological saline solutionother than blood. Typically, the cell suspension comprises whole bloodof a subject at risk of or diagnosed as comprising a tumor. For example,the patient is suspected of having, has been diagnosed as having, or issuspected or diagnosed as having metastatic disease of melanoma, colon,prostate, breast, liver, lung, pancreatic, brain, or blood. CTCs can bepresent before the patient has developed metastatic disease. Therefore,early detection of CTCs is clinically important, because such detectionrepresents an early identification of patients likely to progress todevelop metastatic disease.

Optionally, erythrocyte lysis is carried out as a pretreatment stepprior to flowing cells through the device.

The device is characterized by physical parameters that distinguishtumor cells from non-tumor cells, e.g., normal erythrocytes orleukocytes. For example, the constriction comprises a width from 4 μm-10μm, length of 1 μm-100 μm, and 1-10 constrictions in series. Theestimated speed of the cells can range from 10 mm/s to 10 m/s. To pushor propel the cell suspension through the device, the method furthercomprises applying a pressure to cells. Pressure is used to drive thecell suspension through the device, and the transit through theconstriction point is what deforms the cells and leads to membranedisruption, and therefore delivery.

The method involves introducing into the tumor cell a detectablecompound. Thus, the cell suspension comprises a payload or the methodfurther comprises a step of incubating said cell suspension in thesolution containing a payload for a predetermined time after it passesthrough the constriction. For example, the payload comprises a magneticparticle such as a nanoparticle, a fluorescent particle, such as aquantum dot or carbon nanotube, or a fluorescent dye or protein, orgenetic material (DNA or RNA) that codes for a fluorescent protein orother compound that enables detection (e.g., luciferase). Alternativelyone could deliver a combination of the aforementioned materials toenable detection and simultaneous manipulation of the cells. Forexample, one could deliver a fluorescent particle to enable sorting andco-deliver DNA, RNA or a protein to facilitate subsequent tumor cellsurvival and encourage its growth and proliferation post-sorting toenable further studies of cultured metastatic cells.

Also within the invention is a microfluidic system for distinguishingtumor cells from non-tumor cells, comprising a microfluidic channeldefining a lumen and being configured such that a tumor cell suspendedin a buffer can pass therethrough and is constricted compared to anon-tumor cell. Non tumor cells may be deformed to some extent; however,the key is that the tumor cells are deformed enough to cause a cellmembrane disruption whereas the non-tumor cells are not deformed enoughto result in membrane disruption due to their smaller relative size. Themembranes of smaller cells are not disrupted or disrupted less thanlarger cells, e.g., in some cases, both smaller and larger cells aredisrupted but smaller cells receive less material than the larger cells.The microfluidic channel includes a cell-deforming constriction, whereina diameter of the constriction is a function of the diameter of thecell. The constriction is sized to preferentially deform a tumor cellcompared to a non-tumor cell. This preferential deformation is designedto selectively facilitate the delivery of the target material to tumorcells vs. non tumor cells. Selective delivery enables one to enrich thedesired tumor population through sorting/enrichment methods such as flowcytometery (FACS), micromanipulation, magnetic separation, cell culture.

The method is carried out at physiological temperature, e.g., 37° C.,room temperature, e.g., 20° C., or alternatively, at 0-4° C. In somecases, the latter is preferred, because it can yield better deliveryperformance due to delayed membrane repair and minimize background fromendocytosis by reducing the endocytotic activity of cells. As describedabove, the cell suspension is whole blood or any mammalian cellsuspension in a physiological buffer solution such as phosphate bufferssaline (PBS) or tissue culture media as a delivery buffer. In someexamples, PBS is preferred due to reduced effects from Ca or Mg intissue culture media.

In an aspect, isolating or identifying a cell based on a physicalproperty of the cell can include providing a cell suspension; passingthe suspension through a microfluidic channel that includes aconstriction; passing the cell suspension through the constriction; and,contacting the cell suspension solution with a compound. Theconstriction can be sized to preferentially deform a relatively largercell compared to a relatively smaller cell.

In another aspect, a microfluidic system for distinguishing tumor cellsfrom non-tumor cells can include a microfluidic channel defining a lumenand being configured such that a tumor cell suspended in a buffer canpass therethrough and is constricted compared to a non-tumor cell. Themicrofluidic channel can include a cell-deforming constriction. Adiameter of the constriction can be a function of the diameter of thecell.

One or more of the following features can be included. For example, thephysical property can be one or more of size and diameter. The cellsuspension can include one or more of: peripheral blood cells; and atleast two different cell types having different physical properties. Thecell suspension can include an erythrocyte-depleted population ofperipheral blood cells. The larger cell can include a circulating tumorcell and the smaller cell can include a leukocyte. The compound caninclude a molecular mass of 0.5 kDa to 5 MDa. The compound can include amolecular mass of 3 kDa to 10 kDa. The compound can include a detectablemarker (e.g., quantum dots, cyanine, fluorescein, rhodamine, andderivatives thereof such as fluorescein isothiocyanate (FITC) orTetramethylrhodamine isothiocyanate (TRITC) or NHS-Rhodamine, maleimideactivated fluorophores such as fluorescein-5-maleimide, as well as AlexaFluors), an active biomolecule, and/or a toxin, (e.g., Pseudomonasexotoxin, Diphtheria toxin, and ricin, caspase proteins, antibodies thatinterfere with essential cell functions (e.g. antibodies againsttubulin)) for selectively killing target cells. The compound caninfluence cell function (e.g. transcription factors, siRNA, DNA, mRNA,antibodies, small molecule drugs) and/or can induce cell death. Thecompound can enter the cell after the cell has passed through theconstriction. The suspension can include whole blood. The suspension caninclude whole blood of a subject at risk of or diagnosed as comprising atumor. The tumor can include melanoma, colon, prostate, lung,pancreatic, breast, liver, brain, or blood cancer. The constriction caninclude a width from 4 μm-10 μm, length of 1 μm-100 μm, and 1-10constrictions in series. A speed of the cells traversing a constrictioncan range from 10 mm/s to 10 m/s. A pressure can be applied to the cellsuspension to drive cells through the constriction of a microfluidicchannel.

The cell suspension can include a payload or the cell suspension can beincubated in the solution containing a payload for a predetermined timeafter it passes through the constriction. The payload can include amagnetic particle a fluorescent particle, such as a quantum dot orcarbon nanotube, or a fluorescent dye or protein, or genetic material(DNA or RNA) that codes for a fluorescent protein or other compound thatenables detection (e.g. luciferase).The constriction can be sized topreferentially deform a tumor cell more than a non-tumor cell.

These and other capabilities of the invention, along with the inventionitself, will be more fully understood after a review of the followingfigures, detailed description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a system for size selective tagging of CTCs byrapid mechanical deformation.

FIG. 2 is a bar graph showing that combining size selective delivery ofthe microfluidic platform with antibody staining for CD45 produces asample enrichment factor over an order of magnitude better than eithertechnique independently.

FIG. 3A is a schematic diagram of cell labeling. Red blood cells (RBCs)were depleted from whole blood by RBC lysis using standard erythrocytelysis reagents such as eBioscience RBC lysis buffer (Cat. No. 00-4333).The resulting suspension flowed through the constriction channelmicrofluidics device incubated with a fluorescent dye (and optionallyother compounds). The suspension was then labeled for CD45 and processedon a fluorescence-activated cell sorter (FACS) machine to collect thenon-CD45+ cells that have been labeled with the fluorescent dye.

FIG. 3B is a series of flow cytometry plots of cascade blue conjugated 3kDa dextran delivered by CellSqueeze devices to PBMCs (30-6 chip at 50psi), HT-29 (30-6 chip at 50 psi), SK-MEL-5 (10-7 chip at 50 psi), andPANC-1(10-7 chip at 50 psi).

FIG. 3C is a series of transmitted light and fluorescence micrographs ofPanc-1 tumor cells and blood cells before and after passing through theconstriction channel. The pre-delivery cells are incubated in thepresence of dye to correct for background endocytosis. The post-deliveryimages were taken 24 h after delivery to demonstrate retention of dyeand ability of the cells to adhere and grow following delivery. Althoughlarge blood cells can also get labeled in the process, these datademonstrate selective labeling of tumor cells.

FIG. 4 is a plot of PBMC delivery versus percent PBMC in PBMC andlymphoma mixture showing selective delivery of dyes to lymphoma cellsvs. healthy PBMCs. Even when the suspension is 99.9% healthy PBMCs bynumber, in some implementations up to 8 times specificity in deliverycan be acheived. In other implementations, greater specificity can beachieved.

FIG. 5A is a FACS plot of tetramethylrhodamine dextran-labeledPanc-1-GFP cells spiked into whole blood (40 cells/ml) and processedwith a CD45 counter stain (APC).

FIG. 5B is a FACS plot of GFP versus CD45, demonstrating how PANC-1 GFPtagging could be verified independently based on GFP fluorescence. TheP5 gate would be used as a basis for sorting candidate CTCs, P4 is usedto verify the identity of PANC-1 GFP cells. Green dots are accurate hits(P4 & P5), red dots are false positives (P5 only), blue dots are misses(P4 only).

FIG. 5C is an image of histopathology of HTB1760's primary tumorconfirms pancreatic ductal adenocarcinoma.

DETAILED DESCRIPTION

CTCs are tumor cells that are found in the bloodstream, and are believedto be responsible for the dissemination of cancer to distant organs.CTCs are regarded as minimally-invasive, “liquid biopsies” for cancerpatients and are useful as prognostic indicators for patient outcome andtreatment efficacy. Comprehensive characterizations of these singlecells provide a better understanding of metastatic dissemination,treatment resistance, and tumor biology.

A typical human erythrocyte has a disk diameter of approximately 6.2-8.2μm and a thickness at the thickest point of 2-2.5 μm and a minimumthickness in the center of 0.8-1 μm, being much smaller than most otherhuman cells. Leukocytes (white blood cells) include neutrophils (12-14μm diameter), eosinophils (12-17 μm diameter), basophils (14-16 μmdiameter), lymphocytes (average 6-9 μm in diameter for resting, and10-14 μm diameter for activated), and monocytes, the largest type ofwhite blood cells that can be up to 20 μm in diameter. As shown in FIG.1, the size difference between CTCs and hematologic cells generallypermits distinguishing CTCs from other cells in circulating blood (CTCs·9-20 μm; RBC ˜8 μm discoid; leukocytes ˜7-12 μm). See FIG. 1.Subsequent tumor cell specific labeling using antibodies (orcell-specific fragments thereof) or other tumor cell specific ligandsincrease the selectivity of the method.

Since CTCs are present as one in 10⁶-10⁷ mononuclear cells in thebloodstream, high-sensitivity enrichment techniques are used that relyon immunological or morphological differences in CTCs from the bloodcells. Immunological approaches often target epithelial cell surfacemarkers (such as EpCAM) and tumor-specific proteins (such as Her2-neu,MUCI/MUC2, carcinoembryonic antigen (CEA), mammaglobulin, andalpha-fetoprotein) or aim to deplete CD45+ cells. Microfilters,density-gradient separations, and microfluidics platforms are examplesof morphology-based methods. All of these approaches have inherentbiases, suffer from low enrichment efficiencies and a significant numberof CTCs may down-regulate surface antigens or exhibit varyingmorphological features. These biases pose a significant challenge in thefield as it is still largely unknown which subset of CTCs areresponsible for metastasis or are reliable prognostic markers. Thus, itis important to develop techniques that can ensure high sensitivityisolation of all candidate CTC sub-types to screen for the mostclinically relevant candidates. The devices and methods described hereinpermit the isolation and enumeration of the CTC subtype of interest.

A combined enrichment method integrates both immunological andmorphologic-based approaches to tag and isolate pure CTCs with less biasand based on tunable parameters. The method combines microfluidicintracellular delivery (FIG. 1) and antibody staining to yield robust,high sensitivity purification of circulating tumor cells from wholeblood (FIG. 2) comprises a width from 4 μ-10 μm, length of 1 μm-100 μm,and 1-10 constrictions in series. The estimated speed of the cells canrange from 10 mm/s to 10 m/s. The specific device parameters chosen aredictated by the target tumor cell type, e.g., a different device designis used to select CTCs for a melanoma patient vs. a colon cancerpatient. Examples of tumor cell sizes/diameters include; melanoma ˜15um, colon cancer ˜11 um, and pancreatic cancer ˜15 um.

In this approach, a rapid mechanical deformation delivery systemexploits the inherent size difference between many CTCs and thesurrounding blood cells to selectively deliver fluorescent, magneticand/or other distinguishing materials to the tumor cells. In furtherprocessing, antibody-based fluorescent and/or magnetic tagging is usedto enhance the contrast between the candidate CTCs and the surroundingblood cells. By uniquely combining size-based and immunologicalapproaches to CTC isolation, this technology has demonstrated utilityfor the non-biased isolation of candidate tumor cells from patientsamples for analysis. In some implementations, both smaller and largercells are deformed but the smaller cells membrane is not deformed to thepoint that the membrane becomes compromised. For example, to selectivelydelivering to 15 μm tumor cells in whole blood where most healthy whiteblood cells are ˜8 μm in size, a 6 um width constriction can be used.Such a constriction would deform both cell types but would verypreferentially disrupt the membrane of the 15 μm tumor cells not the 8μm blood cells.

Clinical/Translation Relevance

CTCs are being explored as surrogates for tumor biopsies forunderstanding mechanisms of resistance and guiding the selection oftargeted therapies. Measures of the number and composition of CTCsbefore and after treatment indicate treatment efficacy and prognosis.The approach utilizes a robust, high-throughput, disposable device forthe tagging of CTCs based on cell size and surface antigens. Moreover,the ability to deliver a diversity of macromolecules also enables one todeliver molecular probes (such as antibodies, quantum dots, carbonnanotubes, and molecular beacons) that respond to the intracellularenvironment and thus provide further information on the intracellularproperties of the target cell. This combinatorial approach provides arobust platform capable of enriching CTC populations that would havebeen missed by alternative methods that rely solely on immunological ormorphological separation. The technique is useful to isolate patients'CTCs.

Example 1

Whole blood or other cell suspensions are processed using both unlabeledand/or antibody-coated magnetic beads. These cells are then isolatedusing a high-fidelity, magnetic enrichment system for rare cells. Ananowell technology may also be used to achieve high purity isolationsby imaging and robotically-retrieving single cells of interest from anelastomeric array of 84,672 subnanoliter wells.

Obtaining single, live, pure, intact CTCs of diverse phenotypes allows ahost of characterization efforts from the genomic to functional levelswith immediate clinical and translational relevance. The methods permita highly sensitive and specific enrichment of live, diverse CTCs withreduced bias.

Example 2

Magnetic nanoparticles are delivered to tumor cell lines & PBMCs.Nanoparticle delivery to EpCAM-expressing, epithelial cancer cell lines,e.g., HT-29, LNCaP, and SK-BR-3, is compared to bulk peripheral bloodmononuclear cell (PBMC) suspensions derived from human blood.

10 nm iron-oxide nanoparticles with a polyethylene glycol (PEG) surfacecoating are delivered to cancer cells mixed with whole blood, and theresulting mixture of tagged cells are processed using the cellseparation system described above. For example, the microfluidicdelivery system was used to induce a rapid mechanical deformation of acell to generate transient pores in the cell membrane (FIG. 1). Theapproach has demonstrated an ability to deliver a range of materials,including proteins, RNA, DNA and nanoparticles to a variety of celltypes and works with whole blood, a medium that often poses problems formicrofluidic systems.

Exemplary tagging molecules, e.g., 3 kDa and 70 kDa,fluorescently-labeled, dextran polymers as model molecules, were used todiscriminate between PBMCs and two different cancer cell lines based onsize alone. The results also indicate the utility of the system for theselective delivery of magnetic particles to tumor cells in the blood.PEG coated iron-oxide particles are used to magnetically tag coloncancer (e.g., as exemplified by the cell line HT-29). Further enrichmentis accomplished using conjugation of FITC to the iron-oxide nanoparticlesurface to directly measure nanoparticle uptake.

PEG coated 10 nm iron-oxide nanoparticles are delivered to cellsuspensions that are suspected of containing or are known to containCTCs, e.g., a patient-derived blood sample, or cell lines HT-29, LNCaP,and SK-BR-3 cells, separately mixed with whole blood. The resultingmixture of tagged cells are then purified, e.g., using a high fidelitymagnetic separator. The separator accurately discriminates between themodel CTCs with high iron-oxide content and less- effectively labeledPBMCs. Optionally, red blood cells are lysed prior to treatment,nanoparticle concentration increased, their size altered, orincorporating multiple treatment steps.

Example 3

A combined immunological and morphologic-based method is carried out asfollows. After cell size-based processing by the device, cells aretreated with an antibody or other tumor cell specific ligand such asfluorescently labeled anti-CD45 antibodies. The sensitivity andspecificity of three different separation approaches were compared:: 1)device only 2) anti-CD45 antibody only 3) device+anti-CD45 antibody.Morphologic tagging (device)+immunological tagging (e.g., anti-CD45antibodies) was found to show superior sensitivity (and specificity)relative to either of the individual techniques (FIG. 2). For example, a2-5× increase in sensitivity and/or a 2-5× increase in specificityrelative to anti-CD45 antibodies alone is observed. Enrichment factor ofover an order of magnitude was observed (FIG. 2).

Example 4

In one example, the devices are fabricated out of silicon and glass.Alternatively, the device is fabricated using a polymer such assilicone, PDMS, polycarbonate, acrylic, polypropylene, polystyrene.Either device is sterilized (heat or gamma radiation) and disposable.Performance of the devices is validated for various cell types usingmaterials and parameters. For example, performance at a range of flowspeeds (100 mm/s-10,000 mm/s) using PEG coated quantum dots (rangingfrom 10-50 nm in size) is used to determine if the delivery efficiencyof nanoparticles and cell viability. Exemplary device are described inPCT/US2012/060646, hereby incorporated by reference.

Advantages

When compared to existing approaches this method has the followingadvantages. Relative to antibody-based methods, this approach provides anon-biased isolation procedure that is generalizable to most cancertypes and is independent of any particular cell surface marker. Thedevice and method accomplishes the identification of CTCs that could notbe isolated by existing markers and thus, has significant diagnostic andprognostic implications.

Relative to existing size-based isolation methods, the device andmethods described herein provide far higher throughput and are tunableby varying “W” (FIG. 1) to capture specific CTC size ranges. Forexample, a 6 μm width constriction is suitable for the capture of coloncancer cells whereas a 7 μm, and 8 μm width are suitable for the captureof pancreatic cancer and melanoma cells respectively. Moreover, unlikeexisting technologies, this system is combined with antibody-basedtechnologies to enhance isolation sensitivity and/or enablemulti-parametric isolation of subsets of CTCs (for example by isolatingCTCs of a certain size+surface marker).

By enabling the effective, robust isolation of CTCs from a range ofcancer types this technology would be a valuable platform in the fightagainst cancer. The prognostic and diagnostic potential of thistechnology could enable the identification of new genes that arecritical to cancer progression and thus enable the development of noveltherapeutics. It may also provide a more accurate prediction of patientlife-expectancy and treatment efficacy.

The CTC isolation methods described herein combines immunological andsize-based isolation to yield a high enrichment factor/recovery rate andadjustable bias (marker specific vs. size specific).

Although a few variations have been described in detail above, othermodifications are possible. For example, the implementations describedabove can be directed to various combinations and subcombinations of thedisclosed features and/or combinations and subcombinations of severalfurther features disclosed above. In addition, the logic flows describedherein do not require the particular order described, or sequentialorder, to achieve desirable results. Other embodiments may be within thescope of the following claims.

1. A method for isolating, identifying, or manipulating a cell based ona physical property of said cell comprising: providing a cellsuspension; passing said suspension through a microfluidic channel thatincludes a constriction, said constriction being sized to preferentiallydeliver a compound to a group of cells having a relatively differentphysical property than another group of cells; passing the cellsuspension through the constriction; and contacting said cell suspensionsolution with a compound.
 2. The method of claim 1, wherein the physicalproperty is one or more of size, diameter, and membrane stiffness. 3.The method of claim 1, wherein said cell suspension comprises one ormore of: peripheral blood cells; and at least two different cell typeshaving different physical properties.
 4. The method of claim 1, whereinsaid cell suspension comprises an erythrocyte-depleted population ofperipheral blood cells.
 5. The method of claim 1, wherein said largercell comprises a circulating tumor cell and said smaller cell comprisesa leukocyte.
 6. The method of claim 1, wherein said compound comprises amolecular mass of 0.5 kDa to 5 MDa.
 7. The method of claim 1, whereinsaid compound comprises a molecular mass of 3 kDa to 10 kDa.
 8. Themethod of claim 1, wherein said compound comprises one or more of adetectable marker, an active biomolecule, and a toxin.
 9. The method ofclaim 1, wherein a detectable marker enters said cell after said cellhas passed through said constriction.
 10. The method of claim 1, whereinsaid suspension comprises whole blood.
 11. The method of claim 1,wherein said suspension comprises whole blood of a subject at risk of ordiagnosed as comprising a tumor.
 12. The method of claim 10, whereinsaid tumor comprises melanoma, colon, prostate, lung, pancreatic,breast, liver, brain, or blood cancer
 13. The method of claim 1, whereinsaid constriction comprises a width from 4 μm-10 μm, length of 1 μm-100μm, and 1-10 constrictions in series.
 14. The method of claim 1, whereina speed of the cells traversing a constriction ranges from 10 mm/s to 10m/s.
 15. The method of claim 1, further comprising applying a pressureto the cell suspension to drive cells through the constriction of amicrofluidic channel.
 16. The method of claim 1, wherein said cellsuspension comprises a payload or wherein said method further comprisesa step of incubating said cell suspension in the solution containing apayload for a predetermined time after it passes through theconstriction.
 17. The method of claim 16, wherein said payload comprisesone or more of a magnetic particle, a fluorescent particle, such as aquantum dot or carbon nanotube, a fluorescent dye, a fluorescentprotein, genetic material (DNA or RNA) that codes for a fluorescentprotein, other compound that enables detection (e.g. luciferase), acompound that influences cell function, and a compound that induces celldeath.
 18. A microfluidic system for distinguishing tumor cells fromnon-tumor cells, comprising a microfluidic channel defining a lumen andbeing configured such that a tumor cell suspended in a buffer can passtherethrough and is constricted compared to a non-tumor cell, whereinthe microfluidic channel includes a cell-deforming constriction, whereina diameter of the constriction is a function of the diameter of thecell.
 19. The system of claim 18, wherein said constriction is sized topreferentially deform a tumor cell more than a non-tumor cell.